Optical spectrometers measure the intensity of light at one or more wavelengths to determine certain characteristics of samples, usually liquid samples. The light, usually in the ultraviolet (UV) and/or visible (Vis) wavelength ranges though other wavelength ranges may be used including the near-infrared (NIR) and infrared (IR) wavelengths ranges, is directed through a sample, and the intensity of the output light at the one or more wavelengths is compared to the intensity of the input light at the one or more wavelengths to determine the characteristics of the sample, such as the absorbance, the transmittance, the fluorescence, and/or the reflectance. The measured characteristics provide information about the identity of the components within the sample, their relative concentrations, and possibly other features of the sample. Optical spectrometers are becoming increasingly popular for analysis of small specimens, such as those having a volume of two microliters (μl) or less based on their value in the fields of biotechnology and pharmacology, where specimens often tend to be available in very limited quantities.
In spectroscopy, optical fibers may be used to transmit the light through the sample in order to analyze the composition of the sample. The optical fiber may include a single fiber or a bundle of multiple fibers. Optical fiber typically consists of a transparent core surrounded by a transparent cladding material with a lower index of refraction. The light is kept in the core by total internal reflection causing the fiber to act as a waveguide. There is a maximum angle from the optical fiber axis at which light may enter an entrance face of the optical fiber, propagate in the core of the fiber, and exit an exit face of the optical fiber. The sine of this maximum angle is the numerical aperture (NA) of the optical fiber. As a result of the maximum angle, different distributions of light transmitted into an optical fiber can result in different intensity measurements out of the optical fiber.
The launch spot size is the area of the optical fiber face that is illuminated by the light from the light source. The diameter of the launch spot depends on the size and positioning of the light source and the properties of the optical elements, such as lenses, between the light source and the entrance face of the optical fiber. The angular distribution is the angular extent of the light from the optical light source incident on the entrance face of the optical fiber. The angular distribution also depends on the size and positioning of the light source and the properties of the optical elements between the light source and the entrance face of the optical fiber. As a result, relative to the optical fiber, the light distribution that is created by the light source and any intervening optical elements and that enters the optical fiber can be defined as the statistical distribution of the light in four degrees of freedom, two spatial and two angular.
Multimode optical fiber launch conditions are typically characterized as being underfilled or overfilled. An underfilled optical fiber concentrates most of the optical power in the center of the optical fiber. An underfilled launch results when the launch core diameter and the angular distribution are smaller than that of the optical fiber core.
UV-Vis spectroscopy generally measures the absorption or reflectance of a sample in the ultraviolet-visible spectral region, and thus, uses light in the visible and adjacent (near-UV and NIR) ranges. The absorption or reflectance in the visible range directly affects the perceived color of the chemicals involved. The Beer-Lambert law states that the absorbance of a sample is directly proportional to the concentration of the absorbing species in the sample and the path length. Thus, for a fixed path length, UV/Vis spectroscopy can be used to determine the concentration of the absorber in the sample. It is necessary to know how quickly the absorbance changes with the concentration of the absorber in the sample. As examples, this can be taken from references (tables of molar extinction coefficients), or determined using a calibration curve.
The optical spectrometer measures the intensity of light passing through a sample and compares it to the intensity of light before it passes through the sample. The ratio is called the transmittance. A variety of light sources may be used to perform spectroscopy, such as UV-Vis spectroscopy, though the wavelength of the light source is selected based on the type of components to identify in a sample. For example, the optical spectrometer may utilize a light emitting diode (LED) or a variety of different types of lamps, such as a Tungsten filament, a deuterium arc lamp, and a Xenon arc lamp, etc. as the light source.
The rated lifetime of an LED is given in terms of how long it takes to reach half of its initial intensity. As a result, the fluctuation over the lifetime of the LED is expected to be substantial. In addition, the intensity of the LED may vary as a function of ambient or internal temperature of the instrument with time scales on the order of minutes. Therefore, successive analytical values, measured over a period of minutes to hours, may show noticeable time dependencies that exceed acceptable limits for a useful optical spectrometer. Drift is a change in the reported analytical value of the intensity of the LED over time and may result even when the optical spectrometer is undisturbed. It has been found that different wavelength LEDs may drift at different rates and in different directions and different portions of each LED may themselves drift at different rates with the result that no two optical spectrometers drift identically.
In addition to drift, other factors may cause variation in the intensity values measured by the optical spectrometer. For example, contamination on one or both of the optical fibers may cause variations. Opaque or scattering inclusions in the sample, including dust, particulates, and air bubbles may cause variations. Misplacement of the sample drop on the optical fiber interface, for example due to the normal variation based on manual operation, may cause variations.
The conventional path length for UV-Vis transmission spectroscopy is one centimeter (cm). Thus, there is a 1:1 relationship between absorbance and absorptivity when the latter is reported in units of absorbance per cm. For microliter samples, the path length is much smaller, on the order of 0.005 to 0.02 cm. Thus, absorbance measurements have to be multiplied by large values (50 to 200 times) to report the absolute absorbance in the conventional units expected by users. This factor also multiplies errors in the absorbance measurement making error correction associated with the variations in intensity important design criteria in the manufacture of optical spectrometers, particularly those that utilize an LED light source.